Biology subjects

Crosson, S.

Publications and source records attributed to Crosson, S..

33 records · Page 2Linked to original sources

bioRxiv2020

Flagellar perturbations activate adhesion through two distinct pathways in Caulobacter crescentus

Bacteria carry out sophisticated developmental programs to colonize exogenous surfaces. The rotary flagellum, a dynamic machine that drives motility, is a key regulator of surface colonization. The specific signals recognized by flagella and the pathways by which those signals are transduced to coordinate adhesion remain subjects of debate. Mutations that disrupt flagellar assembly in the dimorphic bacterium Caulobacter crescentus stimulate the production of a polysaccharide adhesin called the holdfast. Using a genome-wide phenotyping approach, we compared surface adhesion profiles in wild-type and flagellar mutant backgrounds of C. crescentus. We identified a diverse set of flagellar mutations that enhance adhesion by inducing a hyper-holdfast phenotype and discovered a second set of mutations that suppress this phenotype. Epistasis analysis of the flagellar signaling suppressor (fss) mutations demonstrated that the flagellum stimulates holdfast production via two genetically distinct pathways. The developmental regulator PleD contributes to holdfast induction in mutants disrupted at both early and late stages of flagellar assembly. Mutants disrupted at late stages of flagellar assembly, which assemble an intact rotor complex, induce holdfast production through an additional process that requires the MotAB stator and its associated diguanylate cyclase, DgcB. We have assigned a subset of the fss genes to either the stator- or pleD-dependent networks and characterized two previously unidentified motility genes that regulate holdfast production via the stator complex. We propose a model through which the flagellum integrates mechanical stimuli into the C. crescentus developmental program to coordinate adhesion. ImportanceUnderstanding how bacteria colonize solid surfaces is of significant clinical, industrial and ecological importance. In this study, we identified genes that are required for Caulobacter crescentus to activate surface attachment in response to signals from a macromolecular machine called the flagellum. Genes involved in transmitting information from the flagellum can be grouped into separate pathways, those that control the C. crescentus morphogenic program and those that are required for flagellar motility. Our results support a model in which a developmental and a mechanical signaling pathway operate in parallel downstream of the flagellum and converge to regulate adhesion. We conclude that the flagellum serves as a signaling hub by integrating internal and external cues to coordinate surface colonization and emphasize the role of signal integration in linking complex sets of environmental stimuli to individual behaviors.

microbiology↗

bioRxiv2020

Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

Perturbations in the early life gut microbiome are associated with increased risk to complex immune disorder like inflammatory bowel diseases. We previously showed maternal antibiotic-induced gut dysbiosis vertically passed to offspring increases experimental colitis risk in IL-10 gene deficient (IL-10-/-) mice. While this could arise from emergence of pathobionts or loss/lack of essential microbes needed for appropriate immunological education, our findings suggest the latter. A dominant Bacteroides strain absent following antibiotic-induced perturbation was cultivated from murine fecal samples. Addition of this strain into mice with antibiotic-induced dysbiosis significantly promoted immune tolerance and reduced incidence of colitis in IL-10-/- mice, but only if engrafted early in life, and not during adulthood. Thus, key members of the gut microbiome are essential for development of immune tolerance to commensal microbes in early life and their addition in presence of gut dysbiosis during this period can reduce colitis risk in genetically prone hosts. HighlightsO_LISpecific gut microbes promote early life immune tolerance to key commensal microbes C_LIO_LILoss of early life keystone microbes increases colitis risk in genetically prone hosts C_LIO_LIEmergence of absent commensal microbes late in life worsened colitis outcome C_LIO_LIEarly life exposure to a missing keystone Bacteroides strain reduced colitis risk C_LI

microbiology↗

bioRxiv2019

Feedback control of a two-component signaling system by a 4Fe-4S-binding receiver domain

Two-component signaling systems (TCSs) function to detect environmental cues and transduce this information into a change in transcription. In its simplest form, TCS-dependent regulation of transcription entails phosphoryl-transfer from a sensory histidine kinase to its cognate DNA-binding receiver protein. However, in certain cases, auxiliary proteins may modulate TCSs in response to secondary environmental cues. Caulobacter crescentus FixT is one such auxiliary regulator. FixT is composed of a single receiver domain and functions as a feedback inhibitor of the FixL-FixJ (FixLJ) TCS, which regulates the transcription of genes involved in adaptation to microaerobiosis. We sought to define the impact of fixT on Caulobacter cell physiology and to understand the molecular mechanism by which FixT represses FixLJ signaling. fixT deletion results in excess production of porphyrins and premature entry into stationary phase, demonstrating the importance of feedback inhibition of the FixLJ signaling system. Although FixT is a receiver domain, it does not affect dephosphorylation of the oxygen-sensor kinase FixL or phosphoryltransfer from FixL to its cognate receiver FixJ. Rather, FixT represses FixLJ signaling by inhibiting the FixL autophosphorylation reaction. We have further identified a 4-cysteine motif in Caulobacter FixT that binds an Fe-S cluster and protects the protein from degradation by the Lon protease. Our data support a model in which oxidation of this Fe-S cluster promotes degradation of FixT in vivo. This proteolytic mechanism facilitates clearance the of the FixT feedback inhibitor from the cell under normoxia and resets the FixLJ system for a future microaerobic signaling event. ImportanceTwo-component signal transduction systems (TCSs) are broadly conserved in the bacterial kingdom and generally contain two molecular components: a sensor histidine kinase and a receiver protein. Sensor histidine kinases alter their phosphorylation state in direct response to a physical or chemical cue, whereas receiver proteins "receive" the phosphoryl group from the kinase to regulate a change in cell physiology. We have discovered that a single-domain receiver protein, FixT, binds an Fe-S cluster and controls Caulobacter heme homeostasis though its function as a negative feedback regulator of the oxygen-sensor kinase, FixL. We provide evidence that the Fe-S cluster protects FixT from Lon-dependent proteolysis in the cell and endows FixT with the ability to function as a second, autonomous oxygen/redox sensor in the FixL-FixJ signaling pathway. This study introduces a novel mechanism of regulated TCS feedback control by an Fe-S-binding receiver domain.

microbiology↗

bioRxiv2019

A carbonic anhydrase pseudogene sensitizes select Brucella lineages to low CO2 partial pressure

Brucella are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for increased CO2 tension to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA. Ancestral single nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that \"rescue\" a functional bcaA reading frame, which enables growth under low CO2 and enhances growth rate in high CO2. Consistent with this result, we show that heterologous expression of functional E. coli carbonic anhydrases enables B. ovis growth in air. Growth of wild-type B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are completely insensitive to CO2 shifts. Wild-type B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in activation of its virB type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.\n\nImportanceBrucella spp. are highly related, but exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require increased CO2 tension to be cultivated in vitro, and harbor conserved insertional mutations in the carbonic anhydrase, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages, and results in a dramatically increased sensitivity to CO2 limitation.

microbiology↗

bioRxiv2019

Regulation of the Erythrobacter litoralis DSM 8509 general stress response by visible light

Extracytoplasmic function (ECF) sigma factors are a major class of environmentally-responsive transcriptional regulators. In Alphaproteobacteria the ECF sigma factor, {sigma}EcfG, activates general stress response (GSR) transcription and protects cells from multiple stressors. A phosphorylation-dependent protein partner switching mechanism, involving HWE/HisKA_2-family histidine kinases, underlies {sigma}EcfG activation. The identity of these sensor kinases and the signals that regulate them remain largely uncharacterized. We have developed the aerobic anoxygenic photoheterotrophic (AAP) bacterium, Erythrobacter litoralis DSM 8509, as a comparative genetic model to investigate GSR regulation. Using this system, we sought to define the contribution of visible light and a photosensory HWE kinase, LovK, to GSR transcription. We identified three HWE kinases that collectively regulate GSR: gsrK and lovK are activators, while gsrP is a repressor. GSR transcription is higher in the dark than light, and the opposing activities of gsrK and gsrP are sufficient to achieve light-dependent differential transcription. In the absence of gsrK and gsrP, lovK alone is sufficient to regulate GSR transcription in response to light. This regulation requires a photochemically active LOV domain in LovK. Our studies establish a role for visible light and HWE kinases in light-dependent regulation of GSR transcription in E. litoralis, an AAP species.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=174 SRC=\"FIGDIR/small/641647v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (19K):\norg.highwire.dtl.DTLVardef@65ed9borg.highwire.dtl.DTLVardef@1d25e65org.highwire.dtl.DTLVardef@333ff9org.highwire.dtl.DTLVardef@ad2cf4_HPS_FORMAT_FIGEXP M_FIG C_FIG ABBREVIATED SUMMARYGeneral stress response (GSR) systems protect bacteria from a diverse range of physical and chemical stressors. We have developed Erythrobacter litoralis as a new genetic model to study GSR in Alphaproteobacteria and show that three HWE-family histidine kinases collectively regulate GSR transcription via {sigma}EcfG. Visible light is a GSR regulatory signal in E. litoralis, and LovK is a blue-light photosensor kinase that functions as a dark activated GSR regulator.

microbiology↗

bioRxiv2019

Composition of the holdfast polysaccharide from Caulobacter crescentus

Surface colonization is central to the lifestyles of many bacteria. Exploiting surface niches requires sophisticated systems for sensing and attaching to solid materials. Caulobacter crescentus synthesizes a polysaccharide-based adhesin known as the holdfast at one of its cell poles, which enables tight attachment to exogenous surfaces. The genes required for holdfast biosynthesis have been analyzed in detail, but an inability to isolate sufficient quantities of holdfast has limited efforts to characterize its composition and structure. In this report we describe a method to extract the holdfast from C. crescentus cultures and present a survey of its carbohydrate content. Glucose, 3-O-methylglucose, mannose, N-acetylglucosamine and xylose were detected in our extracts. Our results suggest that the holdfast contains a 1,4-linked backbone of glucose, mannose, N-acetylglucosamine and xylose that is decorated with branches at the C-6 positions of glucose and mannose. By defining the monosaccharide components in the polysaccharide, our work establishes a framework for characterizing enzymes in the holdfast pathway and provides a broader understanding of how polysaccharide adhesins are built.

microbiology↗

bioRxiv2019

Regulation of bacterial surface attachment by a network of sensory transduction proteins

Bacteria are often attached to surfaces in natural ecosystems. A surface-associated lifestyle can have advantages, but shifts in the physiochemical state of the environment may result in conditions in which attachment has a negative fitness impact. Therefore, bacterial cells employ numerous mechanisms to control the transition from an unattached to a sessile state. The Caulobacter crescentus protein HfiA is a potent developmental inhibitor of the secreted polysaccharide adhesin known as the holdfast, which enables permanent attachment to surfaces. Multiple environmental cues influence expression of hfiA, but mechanisms of hfiA regulation remain largely undefined. Through a forward genetic selection, we have discovered a multi-gene network encoding a suite of two-component system (TCS) proteins and transcription factors that coordinately control hfiA transcription and surface adhesion. The hybrid HWE-family histidine kinase, SkaH, is central among these regulators and forms heteromeric complexes with the kinases, LovK and SpdS. The response regulator SpdR indirectly inhibits hfiA expression by activating two XRE-family transcription factors that directly bind the hfiA promoter to repress its transcription. This study provides evidence for a model in which a consortium of environmental sensors and transcriptional regulators integrate environmental cues at the hfiA promoter to control the attachment decision. Author summaryLiving on a surface within a community of cells confers a number of advantages to a bacterium. However, the transition from a free-living state to a surface-attached lifestyle should be tightly regulated to ensure that cells avoid adhering to toxic or resource-limited niches. Many bacteria build adhesive structures at their surfaces that enable attachment. We sought to discover genes that control development of the Caulobacter crescentus surface adhesin known as the holdfast. Our studies uncovered a network of signal transduction proteins that coordinately control the biosynthesis of the holdfast by regulating transcription of the holdfast inhibitor, hfiA. We conclude that C. crescentus uses a multi-component regulatory system to sense and integrate environmental information to determine whether to attach to a surface, or to remain in an unattached state.

microbiology↗

bioRxiv2019

Brucella EipB is periplasmic β-spiral protein required for envelope stress resistance and infection

The Gram-negative cell envelope is a remarkably diverse structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. We show that a conserved DUF1849-family protein, EipB, is secreted to the periplasmic space of Brucella, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual fourteen-stranded {beta}-spiral structure that contains a conserved disulfide bond. EipB has structural features that resemble the LolA and LolB lipoprotein delivery system, though the overall topology and fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in both cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of B. abortus infection. Transposon disruption of ttpA, which encodes a periplasmic tetratricopeptide repeat (TPR) protein, is synthetically lethal with eipB deletion in B. abortus. ttpA is a known virulence determinant in B. melitensis, and our studies of ttpA deletion and overexpression strains provide evidence that ttpA, like eipB, contributes to cell envelope function in Brucella. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity and to facilitate survival in a mammalian host. ImportanceBrucella species cause brucellosis, a global zoonosis. A gene encoding a conserved uncharacterized protein, EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria. To our knowledge, this study presents the first functional and structural characterization of a protein from the DUF1849 family, to which EipB belongs. EipB is secreted to the periplasm where it forms a spiral-like anti-parallel {beta} structure. Deletion of Brucella eipB results in defects of the cell envelope and in reduced virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.

microbiology↗

bioRxiv2018

A genome-wide analysis of adhesion in Caulobacter crescentus identifies new regulatory and biosynthetic components for holdfast assembly

Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. Caulobacter crescentus produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is a carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We have leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multi-day time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and flagellum control holdfast synthesis separately by modulating the holdfast inhibitor hfiA. This study defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of C. crescentus to surfaces.\n\nImportanceBacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. Caulobacter crescentus produces a structure called the holdfast, which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.

microbiology↗

bioRxiv2018

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded {beta}-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the integrity of the cell envelope. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

microbiology↗

bioRxiv2018

Coherent feedforward regulation of gene expression by Caulobacter σT and GsrN during hyperosmotic stress

GsrN is a conserved small RNA that is under transcriptional control of the general stress sigma factor, {sigma}T, and that functions as a post-transcriptional regulator of Caulobacter crescentus survival under multiple stress conditions. We have defined features of GsrN structure that determine survival under hyperosmotic stress, and have applied transcriptomic and proteomic methods to identify regulatory targets of GsrN under hyperosmotic conditions. The 5 end of GsrN, which includes a conserved cytosine-rich stem loop structure, is necessary for cell survival after osmotic upshock. GsrN both activates and represses gene expression in this stress condition. Expression of an uncharacterized open reading frame predicted to encode a glycine-zipper protein, osrP, is strongly activated by GsrN. Our data support a model in which GsrN physically interacts with osrP mRNA through its 5 C-rich stem loop to enhance OsrP protein expression. We conclude that sigT, gsrN, and osrP form a coherent feedforward loop in which {sigma}T activates gsrN and osrP transcription during stress, and GsrN activates OsrP protein expression at the post-transcriptional level. This study delineates transcriptional and post-transcriptional layers of Caulobacter gene expression control during hyperosmotic stress, uncovers a new regulatory target of GsrN, and defines a coherent feedforward motif in the Caulobacter GSR regulatory network.\n\nImportanceBacteria inhabit diverse niches, and must adapt their physiology to constant environmental fluctuations. A major response to environmental perturbation is to change gene expression. Caulobacter and other alphaproteobacteria initiate a complex gene expression program known as the general stress response (GSR) under conditions including oxidative stress, osmotic stress, and nutrient limitation. The GSR enables cell survival in these environments. Understanding how bacteria survive stress requires that we dissect gene expression responses, such as the GSR, at the molecular level. This study is significant as it defines transcriptional and post-transcriptional layers of gene expression regulation in response to hyperosmotic stress. We further provide evidence that coherent feedforward motifs influence the system properties of the Caulobacter GSR pathway.

microbiology↗

bioRxiv2018

Evaluation of Caulobacter crescentus strain fitness at the genomic scale during growth in natural freshwater

Bacterial genomes evolve in complex ecosystems and are best understood in this natural context, but replicating such conditions in the lab is challenging. We used transposon sequencing to define the fitness consequences of gene disruption in the bacterium Caulobacter crescentus grown in natural freshwater, compared to axenic growth in common laboratory media. Gene disruptions in amino acid and nucleotide biosynthesis pathways and in metabolic substrate transport machinery impaired fitness in both lake water and defined minimal medium relative to complex peptone broth. Fitness in lake water was enhanced by insertions in genes required for flagellum biosynthesis and reduced by insertions in genes involved in biosynthesis of the holdfast surface adhesin. We further uncovered numerous hypothetical and uncharacterized genes for which disruption impaired fitness in lake water, defined minimal medium, or both. At the genome scale, the fitness profile of mutants cultivated in lake water was more similar to that in complex peptone broth than in defined minimal medium. Microfiltration of lake water did not significantly affect the terminal cell density or the fitness profile of the transposon mutant pool, suggesting that Caulobacter does not strongly interact with other microbes in this ecosystem on the measured timescale. Fitness of select mutants with defects in cell surface biosynthesis and environmental sensing were significantly more variable in lake water than in defined medium, presumably owing to day-to-day heterogeneity in the lake environment. This study reveals genetic interactions between Caulobacter and a natural freshwater environment, and provides a new avenue to study gene function in complex ecosystems.

microbiology↗

bioRxiv2018

Molecular control of gene expression by Brucella BaaR, an IclR-family repressor

The Brucella abortus general stress response sigma factor, {sigma}E1, directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection. Characterizing the functions of {sigma}E1 regulated genes therefore contributes to understanding of B. abortus physiology and infection biology. Transcription of the IclR family regulator, Bab2_0215, is indirectly activated by {sigma}E1 but its function remains undefined. We present a structural and functional characterization of Bab2_0215, which we have named Brucella adipic acid activated regulator (BaaR). BaaR adopts a classic IclR-family fold and directly regulates the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and represses transcription. We identified three carboxylic acids (adipic acid tetradecanedioic acid, {varepsilon}-aminocaproic acid) and a lactone ({varepsilon}-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhance transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid requires the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a set of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program regulated downstream of {sigma}E1.

biochemistry↗

bioRxiv2017

Gene network analysis identifies a central post-transcriptional regulator of cellular stress survival

Cells adapt to shifts in their environment by remodeling transcription. Measuring changes in transcription at the genome scale is now routine, but defining the functional significance of individual genes within large gene expression datasets remains a major challenge. We applied a network-based algorithm to interrogate publicly available transcription data to predict genes that serve major functional roles in Caulobacter crescentus stress survival. This approach identified GsrN, a conserved small RNA that is directly controlled by the general stress sigma factor, {sigma}{tau}, and functions as a potent post-transcriptional regulator of survival under multiple stress conditions. GsrN expression is both necessary and sufficient to protect cells from hydrogen peroxide, where it functions by base pairing with the leader of katG mRNA and promoting catalase/peroxidase expression. We conclude that GsrN convenes a post-transcriptional layer of gene expression that serves a central functional role in stress physiology.

microbiology↗

bioRxiv2017

Brucella abortus Induces A Warburg Shift In Host Metabolism That Enhances Intracellular Replication Of The Pathogen

Intracellular bacterial pathogens exploit host cell resources to replicate and survive inside the host. Targeting these host systems is one promising approach to developing novel antimicrobials to treat intracellular infections. We show that human macrophage-like cells infected with Brucella abortus undergo a metabolic shift characterized by attenuated tricarboxylic acid cycle metabolism, reduced amino acid consumption, altered mitochondrial localization, and increased lactate production. This shift to an aerobic glycolytic state resembles the Warburg effect, a change in energy production that is well-described in cancer cells, and also occurs in activated inflammatory cells. B. abortus efficiently uses lactic acid as its sole carbon and energy source and requires the ability to metabolize lactate for normal survival in human macrophage-like cells. We demonstrate that chemical inhibitors of host glycolysis and lactate production do not affect in vitro growth of B. abortus in axenic culture, but decrease its survival in the intracellular niche. Our data support a model in which infection shifts host metabolism to a Warburg-like state, and B. abortus uses this change in metabolism to promote intracellular survival. Pharmacological perturbation of these features of host cell metabolism may be a useful strategy to inhibit infection by intracellular pathogens.\n\nIMPORTANCEBrucella spp. are intracellular bacterial pathogens that cause disease in a range of mammals, including livestock. Transmission from livestock to humans is common and can lead to chronic human disease. Human macrophage-like cells infected with Brucella abortus undergo a Warburg-like metabolic shift to an aerobic glycolytic state where the host cells produce lactic acid and have reduced amino acid catabolism. We provide evidence that the pathogen can exploit this change in host metabolism to support growth and survival in the intracellular niche. Drugs that inhibit this shift in host cell metabolism inhibit intracellular replication and decrease the survival of B. abortus in an in vitro infection model; these drugs may be broadly useful therapeutics for intracellular infections.

microbiology↗